Methods and compositions for high throughput identification of protein/nucleic acid binding pairs

ABSTRACT

Methods and compositions for high-throughput identification of protein/nucleic acid binding pairs are provided. In the subject methods, a nucleic acid probe array, e.g., a molecular beacon probe array, is contacted with a target nucleic acid population to produce a hybridized array. The resultant hybridized array is then contacted with a population of proteins to produce a protein bound array. Any resultant array surface bound target nucleic acid/protein complexes are then detected to identify protein/nucleic acid binding pairs. In certain embodiments, the protein and/or nucleic acid members of the identified protein/nucleic acid binding pairs are further characterized. Also provided are systems and kits for use in practicing the subject methods. The subject invention finds use in a variety of different applications.

FIELD OF THE INVENTION

[0001] The field of this invention is molecular biology, particularlyprotein/nucleic acid binding interactions and protocols for theidentification thereof.

BACKGROUND OF THE INVENTION

[0002] Identification of protein-nucleic acid interactions is paramountin understanding the underlying molecular mechanisms in cellularprocesses such as replication, transcription, and signaling. Oneimportant component in the characterization of DNA/RNA binding proteinsis the analysis of sequence specific interactions using “footprinting”techniques, in which the sequence of the protein binding domain of anucleic acid is identified.

[0003] One footprinting protocol that finds use is based on ligationmediated polymerase chain reaction (LMPCR) (Mueller, P. R. and Wold, B.(1989) Science 246: 780-786). Reagents that are commonly employed inthis protocol include DNasel, DMS (dimethylsulfate) and UV light. Inthese footprinting protocols, a given nucleic acid, typically of knownsequence, is screened for the presence of protein binding sequences bycontacting the nucleic acid with one or more test nucleic acid bindingproteins. Specific sequences along the nucleic acid that are bound tothe protein(s) are protected from nucleophilic attack or cross-linkingby the reagents, thus creating a “footprint” across this region(s) inthe nucleic acid. The protected region is then identified by firstcleaving the DNA at the lesion, and annealing a gene specific primer tothe region of interest. This primer is extended using a processive DNAPolymerase to the cleavage site, creating a blunt end. A unidirectionallinker (staggered) is then attached to the blunt ended molecule usingDNA ligase. The 3′ end of the longer strand of the linker is ligated tothe 5′ end of the genomic DNA. The shorter strand of the linker lacks a5′ phosphate and therefore is not ligated to the extension product. Asecond gene specific primer and a linker specific primer are annealed tothis product, which is now a suitable substrate for a PCR reaction. Onlymolecules that have both sequences (primer 2 sequence and linkersequence) are amplified. A third gene specific primer (labeled) is thenused to sequence the products that can subsequently be visualized on asequencing gel. In this manner, the protein binding sequence of thenucleic acid is identified.

[0004] Terminal Transferase dependent PCR (TDPCR) is a modified LMPCRmethodology that has been devised for studying protein-RNA interactions(Tornaletti, S, and Pfeifer, G (1995) J. Mol. Biol. 249: 714-728; Chen,H-H, et al. (2000) Nucl. Acid Res. 28: 1656-1664). It uses UV light asthe primary source of creating appropriate lesions (intra-strandpyrimidine dimer formation, primarily between thymidines) within theRNA, which inhibit progression of DNA polymerases.

[0005] Although LMPCR and TDPCR are very powerful techniques in mappingprotein-nucleic acid interaction or binding sites, they suffer fromseveral disadvantages that are summarized below. First, in studyingprotein-nucleic acid interactions using LMPCR/TDPCR, one needs to haveprior knowledge of the gene sequence (or transcript) in question inorder to be able to design appropriate gene specific primers foramplification. Second, the LMPCR/TDPCR protocols are labor intensive andoffer considerable challenges to those not well versed in the art.Third, both LMPCR and TDPCR allow analysis of protein-nucleic acidinteractions at the nucleotide resolution by revealing the footprintthat the protein leaves behind on the nucleic acid. However, they arenot useful techniques in determining the underlying identity of theprotein(s) resulting in such a footprint. To identify the proteins perse, one has to resort to the use of monoclonal antibody protocols, whichsuffer from the drawback that a priori knowledge about the identity ofthe proteins is needed. Because of the above limitations, none of thecurrently employed techniques for identifying protein/nucleic acidbinding pairs can be adopted for high throughput mapping ofsite-specific protein binding sequences.

[0006] As such, there is a continued interest in the development of newprotocols for identifying protein/nucleic acid binding pairs, where thedevelopment of a protocol that could be adapted to a high throughputformat is of particular interest.

[0007] Relevant Literature

[0008] U.S. Patents of interest include: U.S. Pat. Nos. 5,925,517;6,150,097; 6,355,421. Also of interest is: Tyagi & Kramer, NatBiotechnol (1996 Mar) 14(3): 303-8.

SUMMARY OF THE INVENTION

[0009] Methods and compositions for identifying protein/nucleic acidbinding pairs are provided. In the subject methods, a nucleic acid probearray is first contacted with a target nucleic acid population toproduce a hybridized array. The resultant hybridized array is thencontacted with a population of proteins to produce a protein boundarray. Protein/nucleic acid binding pairs are then detected on the arraysurface. In certain embodiments, the protein and/or nucleic acid membersof the identified protein/nucleic acid binding pairs are furthercharacterized.

[0010] In many embodiments, the array employed is a molecular beaconarray having a plurality of distinct molecular beacon probes all labeledwith the same first fluorescent label. In these embodiments, themolecular beacon array is first contacted with a target nucleic acidpopulation to produce a hybridized array. The resultant hybridized arrayis then contacted with a population of proteins all labeled with thesame second fluorescent label to produce a protein bound array. Afeature of the methods of this embodiment is that the first and secondfluorescent labels make up a FRET pair. Any FRET generated signals fromthe resultant protein bound array are then detected from the proteinbound array to identify protein/nucleic acid binding pairs.

[0011] Also provided are systems and kits for use in practicing thesubject methods. The subject invention finds use in a variety ofdifferent applications.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 provides a view of a representative molecular beacon probeof the molecular beacon arrays employed in certain embodiments of thesubject invention.

[0013]FIG. 2 provides an illustration of the hybridization of a targetnucleic acid to a molecular beacon probe and the consequentconformational change of the molecular beacon probe to provide for adetectable signal.

[0014]FIG. 3 provides an illustration of a protein bound to a targetnucleic acid of a molecular beacon array, where the label of the proteinand the label of the molecular beacon are in a FRET relationship.

[0015]FIG. 4 provides an illustration of the effect of distance on theFRET relationship that can be established by the subject methods.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0016] Methods and compositions for high-throughput identification ofprotein/nucleic acid binding pairs are provided. In the subject methods,a nucleic acid probe array, e.g., a molecular beacon probe array, iscontacted with a target nucleic acid population to produce a hybridizedarray. The resultant hybridized array is then contacted with apopulation of proteins to produce a protein bound array. Any resultantarray surface bound target nucleic acid/protein complexes are thendetected to identify protein/nucleic acid binding pairs. In certainembodiments, the protein and/or nucleic acid members of the identifiedprotein/nucleic acid binding pairs are further characterized. Alsoprovided are systems and kits for use in practicing the subject methods.The subject invention finds use in a variety of different applications.

[0017] Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0018] In this specification and the appended claims, the singular forms“a,” “an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0019] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0020] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0021] All publications mentioned herein are incorporated herein byreference for the purpose of describing various invention componentsthat are described in the publications, which might be used inconnection with the presently described invention.

[0022] As summarized above, the subject invention provides methods andcompositions for the high-throughput identification of protein/nucleicacid binding pairs. In further describing the subject invention, themethods will be described first in greater detail, followed by a reviewof the systems and kits provided by the invention for practicing thesubject methods.

[0023] As summarized above, the subject invention provides methods forthe high-throughput identification of protein/nucleic acid bindingpairs. More specifically, the subject invention provides methods ofidentifying protein/nucleic acid binding pairs that exist in a union ofa first set of one or more nucleic acids and a second set of one or moreproteins. The nucleic acid member of the identified binding pairs may beRNA, e.g., cRNA, mRNA etc., or DNA, e.g., single stranded or doublestranded DNA. As such, RNA/protein binding pairs and DNA/protein bindingpairs may be identified by using the subject invention. The sets ofnucleic acids and proteins that are screened or assayed according to thesubject methods may be obtained from a variety of sources, includingnaturally occurring or synthetic sources. In addition, the sources ofthe proteins and nucleic acids that make up the assayed sets may be thesame or different.

[0024] In practicing the subject methods, a nucleic acid probe array isemployed to assay the union of a set of nucleic acids and proteins forthe presence of protein/nucleic acid binding pairs in the union. Toassay the union of the nucleic acid and protein sets, the nucleic acidprobe array is first contacted with a population of target nucleicacids, i.e., the set of target nucleic acids, to produce a hybridizedarray. The resultant hybridized array is then contacted with apopulation of labeled proteins, i.e., the set of proteins, to produce aprotein bound array. The resultant protein bound array is then assayedto detect any resultant surface bound labeled protein molecules in orderto detect protein/nucleic acid binding pairs that exist in the union ofthe assayed nucleic acid and protein sets. Depending on the particularembodiment, the labeling protocol employed to detect the surface boundprotein/nucleic acid complex may vary. Representative labeling protocolsinclude those that employ directly detectable labels and those thatemploy indirectly detectable labels, where the latter of which arecharacterized by having two or more signal producing system members thatwork in concert to produce a detectable signal. Examples of directlydetectable labels include isotopic labels, fluorescent labels, FETlabeling systems, including FRET labeling systems, etc. Examples ofindirectly detectable labels include those found in enzymatic signalproducing systems, e.g., chemillumninescent systems, etc.

[0025] Because of the ease of use and detection of fluorescent labels,in many embodiments employed the labels employed are directly detectablefluorescent labels. Fluorescent labeling systems of interest include FETlabeling systems, where energy transfer between donor and acceptormoieties occurs, where the acceptor may be a second fluorescer, e.g., asis present in FRET systems, or may be a quencher moiety. In certainembodiments of particular interest, a FRET labeling system is employed,where at least two of three main assay components (i.e., the array boundprobe nucleic acids, the target nucleic acids, and the proteins) havelabels that form a FRET pair, and in certain embodiments all three ofthese components have labels that form a FRET pair.

[0026] In many embodiments of particular interest, the array of probenucleic acids that is employed is one that is a molecular beacon array.Since these embodiments are of particular interest, the invention willnow be further described in terms of these embodiments.

[0027] In practicing the subject methods of these embodiments ofparticular interest, a molecular beacon array is employed to assay theunion of a set of nucleic acids and proteins for the presence ofprotein/nucleic acid binding pairs in the union. To assay the union ofthe nucleic acid and protein sets, the molecular beacon array is firstcontacted with a population of target nucleic acids, i.e., the set oftarget nucleic acids, to produce a hybridized array. The resultanthybridized array is then contacted with a population of fluorescentlylabeled proteins, i.e., the set of proteins, to produce a protein boundarray. The resultant protein bound array is then assayed for anyresultant FRET generated signals, which signals are then detected inorder to detect protein/nucleic acid binding pairs that exist in theunion of the assayed nucleic acid and protein sets.

[0028] Molecular Beacon Array

[0029] As such, the first step in the subject methods is to provide amolecular beacon array for use in the subject methods. The molecularbeacon array is a composition of matter that includes a substrate thatdisplays at least one molecular beacon probe immobilized on a surfacethereof, where the arrays employed in the subject invention typicallyinclude a plurality of distinct molecular beacon probes immobilized on asurface of a substrate, where each member of the plurality differs interms of probe sequence, as described in greater detail below.

[0030] The molecular beacon probes of the subject arrays areconformationally labeled probe structures that generate a differentfluorescent signal depending on whether or not they are hybridized to atarget nucleic acid. In other words, the molecular beacon probes areprobes that generate a first fluorescent signal, e.g., a quenchedsignal, undetectable signal, when not hybridized to a target nucleicacid and a second fluorescent signal, e.g., an unquenched fluorescentsignal, when hybridized to a target nucleic acid. While in principle anyconformational probe that functions as described above may be employed,in many embodiments the probes have a molecular beacon structure.

[0031] Molecular beacon conformational probe structures are known tothose of skill in the art and reviewed in, among other places, U.S. Pat.Nos. 5,925,517; 6,150,097 and 6,355,421 (the disclosures of which areherein incorporated by reference); as well as Tyagi & Kramer, NatBiotechnol (1996 Mar) 14(3):303-8. Molecular beacons are single strandednucleic acid or nucleic acid mimetic (e.g., PNA) probes that form astem-loop structure. A fluorophore, i.e., first fluorescent label, andquencher are linked to opposite ends of the molecule. Fluorescence isquenched when the probe is in the stem-loop conformation. However, whenthe probe sequence in the loop anneals to a complementary nucleic acidtarget sequence, the duplex formed overcomes the shorter hairpin-stem sothat the probe undergoes a conformational transition that separates thefluorophore and quencher, such that the signal generated by the firstfluorescent label upon excitation is no longer quenched. FIGS. 1 and 2provide a depiction of a representative molecular beacon probe in thetwo different conformations.

[0032] In the molecular beacon probes employed on the subject molecularbeacon probe arrays, the probe sequence of the stem-loop structure isdesigned to hybridize to at least a portion of a target nucleic acidsequence. The probe sequence length may be any convenient length. Inmany embodiments, the length typically ranges from about 5 about 200residues, e.g., nt, PNA subunits, etc. Often, the probing nucleobasesequence will be 5 to 150 nt in length, e.g., 10 to 100 nt in length,such as 50, 60, 70 nt in length, etc.

[0033] Flanking either side of the probe sequences in the molecularbeacon probes are arm segments. The arm segments are designed to annealto each other and thereby stabilize the interactions that fix the energytransfer of linked donor and acceptor moieties, i.e., first fluorescentlabel and quencher therefore, until the molecular beacon probehybridizes to the target sequence. The arm segments may be of differentlengths, but are typically the same length. The preferred length of thearm segments will depend on the stability desired for the interactions.However, the arm segments must not be so long that they prohibithybridization to the target sequence. Often, the arm segments are fromabout 2 to about 10 subunits in length and more often from about 2 toabout 5 subunits in length. In certain embodiments, both arm segmentsare external to the probing sequence.

[0034] Each molecular beacon probe is labeled such that the probe yieldsa quenched or unquenched fluorescent signal, depending on theconformation of the molecular beacon probe. The labels attached to theprobes comprise a set of energy transfer moieties comprising at leastone energy donor and at least one energy acceptor moiety. Typically, theset includes a single donor moiety and a single acceptor moiety.Nevertheless, a set may contain more than one donor moiety and/or morethan one acceptor moiety. The donor and acceptor moieties operate suchthat the acceptor moiety accepts energy transferred from the donormoiety, resulting in quenching of the signal from the acceptor moiety.

[0035] In many embodiments, the donor moiety is a fluorophore.Representative fluorophores are derivatives of fluorescein, derivativesof bodipy, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS),derivatives of rhodamine, cyanine dyes, e.g., Cy2, Cy3, Cy 3.5, Cy5,Cy5.5, texas red and its derivatives, etc. Though the previously listedfluorophores might also operate as acceptors, in certain embodiments theacceptor moiety is a quencher moiety, e.g., a non-fluorescent aromaticor heteroaromatic moiety, e.g., 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl), etc.

[0036] Transfer of energy from the donor, e.g., first fluorescent label,may occur through collision of the closely associated moieties of a setor through a nonradiative process such as fluorescence resonance energytransfer (FRET). For FRET to occur, transfer of energy between donor andacceptor moieties of a set requires that the moieties be close in space(e.g., less than about 100 Å, often less than about 80 Å) and that theemission spectrum of a donor(s) have substantial overlap with theabsorption spectrum of the acceptor(s). Alternatively, collisionmediated (radiationless) energy transfer may occur between very closelyassociated donor and acceptor moieties whether or not the emissionspectrum of a donor moiety(ies) has a substantial overlap with theabsorption spectrum of the acceptor moiety(ies). This process isreferred to as intramolecular collision since it is believed thatquenching is caused by the direct contact of the donor and acceptormoieties.

[0037] The molecular beacon probes are generally polymeric and may benucleic acids, polymeric mimetics thereof, e.g., PNAs, or copolymers ofnucleotide and non nucleotide residues, e.g., block copolymers ofnucleic acids and nulceic acid mimetics, such as PNAs. The nature of themolecular beacon probes may vary, so long as that function as describedabove.

[0038] As indicated above, in many embodiments an array of theabove-described molecular beacon probes is employed. The molecularbeacon probe arrays include at least two distinct molecular beaconprobes that differ from each other with respect to their probingsequence, and yet are labeled with the same first fluorescent label,e.g., donor label, as described above. The molecular beacon probes ofthe array are immobilized. on e.g., covalently (such as cross-linked ordirectly synthesized through phosphoramidite linkage chemistry) ornon-covalently (such as through biotin/avidin binding pair) attached to,different and known locations on the substrate surface. The probes maybe attached to the surface directly, or through a suitable spacer group,as is known in the array art. Each distinct molecular beacon probe ofthe array is typically present as a composition of multiple copies ofthe probe on the substrate surface, e.g., as a spot or feature on thesurface of the substrate. The number of distinct probes, and hence spotsor similar structures, present on the array may vary, but is generallyat least 1000, and may be as high as 25,000 or higher. The spots ofdistinct probes present on the array surface are generally present as apattern, where the pattern may be in the form of organized rows andcolumns of spots, e.g. a grid of spots, across the substrate surface, aseries of curvilinear rows across the substrate surface, e.g. a seriesof concentric circles or semi-circles of spots, and the like. Thedensity of spots present on the array surface may vary, but willgenerally be at least about 10 and usually at least about 100 spots/cm²,where the density may be as high as 10⁶ or higher, and in certainembodiments will generally not exceed about 10⁵ spots/cm². A variety ofdifferent array configurations and formats, including choice ofsubstrate material, organization of probes, dimensions, etc., are knownand have been developed, where any convenient configuration may beemployed. Representative configurations of interest include, but are notlimited to, those described in U.S. Pat. Nos. 6,372,483; 6,355,421;6,323,043; 6,306,599; 6,242,266; 6,222,030; 6,221,653; 6,180,351;6,171,797; and 6,077,674; the disclosures of which are hereinincorporated by reference.

[0039] In certain embodiments, two or more distinct probes on the arrayform a set of probes that all hybridize to the same target nucleic acid,where the probe sequences of the different members of the set eachhybridize to different domains or regions of the same target nucleicacid. See e.g., FIG. 4, where two probes that hybridize to the sametarget nucleic acid at different locations are illustrated. In certainembodiments, the arrays include sets of molecular probes that span theentire length of a target nucleic acid, such that the entire sequence ofthe target nucleic acid is represented among the different molecularbeacon probes of the set that all hybridize to that target nucleicacid—in other words, a “tiled” set of molecular beacon probes isprovided for a target nucleic acid. Such embodiments find use in.applications where characterization of the cognate sequence of anidentified protein/DNA binding pair is desired, as described more fullybelow.

[0040] Target Nucleic Acid Hybridization

[0041] The next step in the subject methods is to bind the solid supportbound molecular beacon probe(s), e.g., molecular beacon array, with oneor more target nucleic acids under hybridization conditions to produce ahybridized array. In the broadest sense, the target nucleic acid(s)contacted with the array in this step is any nucleic acid, which is tobe screened or assayed together with a protein set to identify whetherit is part of a protein/nucleic acid binding pair. As such, the length,chemical nature and source of the target nucleic acid(s) may varygreatly, depending on the particular protocol being performed. Thenucleic acids may be oligonucleotides, polynucleotides etc. The nucleicacid may be RNA, e.g., cRNA, mRNA, etc., or DNA, including either singlestranded or double stranded DNA, e.g., cDNA, etc.

[0042] In many embodiments, a plurality of distinct nucleic acids arecontacted with the molecular beacon array, e.g., 5 different, 50different, 100 different, 500 different, 1000 different, 10,000different, etc., nucleic acids of differing sequence.

[0043] The plurality of target nucleic acids that is contacted with themolecular beacon array may be generated using any convenient targetnucleic acid generation protocol, where representative target generationprotocols include both linear and geometric amplification protocols,where the generated target nucleic acids may be DNA, RNA etc. In manyprotocols known to those of skill in the art, an initial nucleic acidbiological source is employed, e.g., a cellular or tissue nuclearsource. Any convenient nucleic acid source may be employed.

[0044] A representative protocol of particular interest in certainembodiments includes the linear amplification protocol described in U.S.Pat. No. 6,132,997, the disclosure of which is herein incorporated byreference.

[0045] In many embodiments, the protocol that is employed is one thatgenerates unlabeled target nucleic acids, as a label element on thetarget nucleic acid is not employed and could, potentially though notnecessarily, interfere with the signal producing system that isemployed. If, however, the target nucleic acid is labeled, it is labeledwith a moiety that does not adversely affect the signal producing systememployed in the subject methods, as described in greater detail below.

[0046] Once generated, the population of target nucleic acids iscontacted with the molecular beacon array under hybridization conditionsto produce a hybridized array. In many embodiments, the hybridizationconditions under which contact of the array and the target nucleic acidstakes place are stringent hybridization conditions. The term “stringenthybridization conditions” as used herein refers to conditions that arecompatible to produce duplexes on an array surface between complementarybinding members, i.e., between probes and complementary targets in asample, e.g., duplexes of nucleic acid probes, such as DNA probes, andtheir corresponding nucleic acid targets that are present in the sample,e.g., their corresponding cRNA analytes present in the sample. Anexample of stringent hybridization conditions is hybridization at 37° C.or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate).Another example of stringent hybridization conditions is incubation at42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCI, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10%dextran sulfate, followed by washing in 0.5×SSC with 0.01% SDS followedby another wash of 0.06×SSC at about 65° C. Stringent hybridizationconditions are hybridization conditions that are at least as stringentas the above representative conditions, where conditions are consideredto be at least as stringent if they are at least about 80% as stringent,typically at least about 90% as stringent as the above specificstringent conditions. Other stringent hybridization conditions are knownin the art and may also be employed, as appropriate.

[0047] Contact/binding of the target nucleic acid population with themolecular beacon array as described above results in the production of ahybridized array. As such, duplex nucleic acid structures are producedat any location where a target nucleic acid has hybridized to the probesequence of a surface bound molecular beacon probe. At these locationsof the array, the hybridization of the target nucleic acid to the proberesults in a conformational change of the probe, as illustrated in FIG.2.

[0048] Optionally, following production of the target hybridized nucleicacid array, the hybridized array may be scanned or read, e.g., usingconventional fluorescence detection techniques as described in greaterdetail below, to identify the target nucleic acids present in thecontacted target nucleic acid population.

[0049] Protein Binding

[0050] Following production of the hybridized molecular beacon array,and any signal detection step, e.g., fluorescence scanning step (asmentioned above and described in greater detail below), the hybridizedarray is contacted with at least one labeled protein. A feature of thelabeled protein is that it includes a second fluorescent label which,together with the first fluorescent label of the surface bound molecularbeacon, produces or makes up a FRET pair. Two fluorescent labels areviewed as being a FRET pair for purposes of the present invention if,when positioned sufficiently close to each other (typically less thanabout 100 Å, and usually less than about 80 Å), they participate influorescence resonance energy transfer, such that excitation of one ofthe labels gives rise to emission from the other of the two labels. Avariety of FRET pairs of fluorescent labels are known to those of skillin the art and may be employed. The energy donors of the pairs willgenerally be compounds which absorb in the range of about 300 to about800 nm, more usually in the range of about 450 to about 700 nm, and arecapable of transferring energy to an acceptor fluorophore, whichgenerally absorbs light of a wavelength 15 nm, more usually 20 nm orhigher, than the absorption wavelength of the donor. The acceptor willgenerally emit in the range of about 400 to about 900 nm. Fluorophoresof interest include, but are not limited to: fluorescein dyes (e.g.,5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET), 2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), and2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE)), cyanine dyessuch as Cy5 and Cy3, dansyl derivatives, rhodamine dyes (e.g.,tetramethyl-6-carboxyrhodamine (TAMRA), andtetrapropano-6-carboxyrhodamine (ROX)), DABSYL, DABCYL, anthraquinone,nitrothiazole, and nitroimidazole compounds, and the like. Fluorophoresof interest are further described in WO 01/42505 and WO 01/86001, aswell as the priority U.S. Applications of these documents, thedisclosures of the latter of which are herein incorporated by reference.

[0051] Any convenient protocol may be employed to produce the labeledprotein, as described above. In certain embodiments, the protein ofinterest is labeled with functionalized label reagent that covalentlybonds to the protein and, in doing so, labels the protein. In theseembodiments, the protein is contacted with functionalized label underconditions sufficient for a functional moiety of the protein, e.g., anamine or hydroxyl group, to react with the corresponding functionalmoiety present on the label to produce a covalent bond between the labeland the analyte. As such, functionalized labels employed in theseembodiments of the subject methods include a functional moiety and alabel moiety. The functional moiety of the functionalized labels mayvary greatly, and is chosen in view of the functional moiety present onthe protein to be labeled, e.g., amine groups on the protein. In otherwords, the functional moiety present on the functionalized label is onethat reacts with the functional moiety present on the protein to producea covalent bond between the protein and the label. Representativefunctional moieties that may be present on the label include: amino,sulfhydryl, sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate,isocyanate, anhydride, monochlorotriazine, dichlorotriazine, mono-ordihalogen substituted pyridine, mono- or disubstituted diazine,maleimide, aziridine, sulfonyl halide, acid halide, alkyl halide, arylhalide, alkylsulfonate, N-hydroxysuccinimide ester, imido ester,hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-propionamide,glyoxal, aldehyde, iodoacetyl, cyanomethyl ester, p-nitrophenyl ester,o-nitrophenyl ester, hydroxypyridine ester, carbonyl imidazole, and thelike.

[0052] In many embodiments, the hybridized array in this step incontacted with a population of different proteins, i.e., a protein set,where the proteins are all labeled with the same second fluorescentlabel. By population of different proteins is meant a plurality ofproteins that differ from each other in terms of amino acid sequence,where the number of distinct or different proteins in the population isat least 2, usually at least 50, more usually at least 100, and often ismuch greater, e.g., at least about 500, at least about 1000, at leastabout 2000, at least about 5000 etc.

[0053] In many embodiments, the population of labeled proteins isproduced by contacting an initial source of a plurality of differentproteins with functionalized label, as described above. The initialsource of different proteins may be any convenient source, e.g., asynthetic source, a naturally occurring source, e.g., a cell lysate,tissue homogenate, etc.

[0054] At least one fluorescently labeled-protein, i.e., the proteinset, as described above, is contacted with the hybridized array underprotein/nucleic acid binding conditions sufficient to produce a proteinbound array. Contact may occur using any convenient protocol. As such, afluid sample that includes the at least one fluorescently labeledprotein may be applied to the substrate surface, flowed across thesubstrate surface, or the substrate surface may be immersed in the fluidsample, etc.

[0055] Binding/contact between the surface and sample including the atleast one labeled protein is maintained for a period of time sufficientfor binding between the protein and any recognized nucleic acid bindingsequences present on the substrate surface to occur. As such, thesubstrate surface and the sample are incubated for a period of time andunder conditions sufficient for binding between nucleic acids andproteins of a given protein/nucleic acid binding pair to occur. Thesample and substrate are typically incubated for a period of timeranging from about 5 min to 2 hours, usually from about 15 min to 2hours and more usually from about 30 min to 1 hour. The temperatureduring this incubation period generally ranges from about 0 to about 37°C. usually from about 15 to 30° C. and more usually from about 18 to 25°C. Where desired, the substrate and sample may be agitated duringincubation, e.g., by shaking, stirring, etc.

[0056] The above contacting/incubating steps result in the production ofa protein bound array, which includes one or more surface boundprotein/nucleic acid binding pairs, if such pairs exist in thecollection or union of target nucleic acid and labeled protein sets thatare assayed according to the subject methods. The surface boundprotein/nucleic acid binding pairs may have a structure as illustratedin FIG. 3.

[0057] FRET Signal Detection

[0058] Following production of the protein bound array, the surface ofthe array is assayed for the presence of FRET generated signal. Anyconvenient protocol for detecting FRET generated signal on the surfacemay be employed. Typically, this step involves irradiating the surfacewith a wavelength suitable for absorption of one of the fluorescentlabels so that a FRET generated emission from the other of thefluorescent labels is produced, followed by detection of this FRETgenerated signal. Any convenient protocol for irradiating at the firstwavelength and detecting the FRET emitted signal may be employed. Assuch, reading of the array may be accomplished by illuminating the arrayand reading the location and intensity of resulting fluorescence at eachfeature of the array to detect any protein/nucleic acid bindingcomplexes on the surface of the array. For example, a scanner may beused for this purpose, which is similar to the AGILENT MICROARRAYSCANNER scanner available from Agilent Technologies, Palo Alto, Calif.Other suitable apparatus and methods are described in U.S. patentapplications: Ser. No. 09/846125 “Reading Multi-Featured Arrays” byDorsel et al.; and Ser. No. 09/430214 “Interrogating Multi-FeaturedArrays” by Dorsel et al. These references are incorporated herein byreference.

[0059] Any detected FRET generated signals are then attributed to thepresence of a protein/nucleic acid binding pair at the location of thesurface from which the signal is generated. In this manner, detection ofa FRET generated signal on the surface of the array is employed todetect a protein/nucleic acid binding pair on the surface of the array.

[0060] In certain embodiments, the hybridized array is contacted withtwo distinct populations of labeled proteins, which are differentiallylabeled. By differentially labeled is meant that the two populations arelabeled with different fluorescent labels that are distinguishable fromeach other, e.g., upon excitation they emit at different maxima.Although the two populations are differentially labeled, the label ofthe first population and the label of the second population mustnonetheless form a FRET pair with the first fluorescent label of thesurface bound molecular beacon. In such embodiments, the two differentprotein populations are generally contacted/bound in known amountsrelative to each other with the array, such that the ratio of amounts offirst and second populations contacted/bound to the array is known. Incertain embodiments, substantially equimolar, including equimolar,amounts of the first and second protein populations are contacted/boundwith the array. Embodiments where two differentially labeled proteinpopulations are bound with the hybridized array include applicationswhere the identified protein/nucleic acid binding pairs are to bequantitated, where protein populations are to be compared, e.g.,normal/control pairs; disease/normal pairs, etc.

[0061] Results from the reading may be raw results (such as fluorescenceintensity readings for each feature in one or more color channels) ormay be processed results such as obtained by rejecting a reading for afeature which is below a predetermined threshold and/or formingconclusions based on the pattern read from the array (such as whether ornot a particular target sequence may have been present in the sample).The results of the reading (processed or not) may be forwarded (such asby communication) to a remote location if desired, and received therefor further use (such as further processing). In certain embodiments,the subject methods include a step of transmitting data from at leastone of the detecting and deriving steps, as described above, to a remotelocation. By “remote location” is meant a location other than thelocation at which the array is present and hybridization and/or proteinbinding occurs. For example, a remote location could be another location(e.g. office, lab, etc.) in the same city, another location in adifferent city, another location in a different state, another locationin a different country, etc. As such, when one item is indicated asbeing “remote” from another, what is meant is that the two items are atleast in different buildings, and may be at least one mile, ten miles,or at least one hundred miles apart. “Communicating” information meanstransmitting the data representing that information as electricalsignals over a suitable communication channel (for example, a private orpublic network). “Forwarding” an item refers to any means of gettingthat item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. The data may be transmittedto the remote location for further evaluation and/or use. Any convenienttelecommunications means may be employed for transmitting the data,e.g., facsimile, modem, internet, etc.

[0062] Identification of Protein/Nucleic Acid Binding Pairs

[0063] As indicated above, the data generated upon reading of the arrayis employed to identify protein/nucleic acid binding pairs that exist inthe union of the set of proteins and nucleic acids that are assayed withthe array according to the subject methods. More specifically, the data,i.e., FRET generated signal, is employed to identify protein/nucleicacid binding pairs that exist in the combined set of target nucleicacids and labeled proteins that are contacted with the molecular beaconarray during practice of the subject methods. For example, where thetarget nucleic acid population and the labeled protein populationcontacted with the molecular beacon array are obtained from the samecellular/tissue source, any observed FRET generated signals indicate thepresence of protein/nucleic acid binding pairs found in the cell/tissuesource. In other embodiments where the target nucleic acid and proteinpopulations are from different sources, FRET generated signals indicateprotein/nucleic acid binding pairs present in the union of the two setsfrom different sources. As such, the array is scanned for the presenceof FRET generated signals, where any observed signals indicate thepresence of a protein/nucleic acid binding pair and therefore can berelated to the presence of a protein/nucleic acid binding pair, i.e.,the presence of a protein/nucleic acid binding pair can be derived fromthe observed FRET generated signal.

[0064] Optional Additional Steps

[0065] Following identification of the any protein/nucleic acid bindingpairs, the identified protein/nucleic acid binding pairs may be furtheranalyzed, e.g., to identify the nature of the protein member and/ornucleic acid member of the pair.

[0066] Protein Identification

[0067] The protein member of the protein/nucleic acid binding pair maybe further characterized/identified using a number of differentprotocols, including protocols known to those of skill in the art.Basically, any convenient protocol may be employed, where the protocolyields additional information with regard to the nature/identity of theprotein member of the identified protein/nucleic acid binding pair. Onerepresentative protein characterization protocol that may be employed isto produce an enzyme digest profile for the protein, where the proteinis then compared to a reference database of digest profiles to identifythe protein. For example, the protein member of the protein/nucleic acidbinding pair may be digested with trypsin to produce a trypsin digest,where the resultant fragments are analyzed by tandem MALDI-TOF/ESI massspectrometry to produce a searchable profile. Identification of theprotein is then done by comparing the resultant profile to a database ofreference profiles generated by a theoretical trypsin digest createdagainst all available protein sequences in a given protein sequencedatabase (for example, SWISS-PROT). Such a protocol is described in:Gygi, S. P et al. Nat. Biotech. (1999), Vol 17: 994-999, and Griffin, T.J. et al. Anal. Chem. (2001), Vol 73: 978-986. Other proteincharacterization protocols that may be employed include, but are notlimited to: yeast two-hybrid protocols, protein fragment complementationassay protocols; and the like.

[0068] Where desirable, larger amounts of the protein member of theprotein/nucleic acid binding pair may be obtained prior tocharacterization. Any convenient protocol for obtaining larger amountsof the to be characterized protein member may be employed. For example,one may be use the nucleic acid member of the pair to purify additionalprotein from the original source employed in the methods, as describedabove. For example, the nucleic acid member of the identifiedprotein/nucleic acid binding pair may be amplified to produce solidphase capturable nucleic acids, e.g., the nucleic acid may be amplifiedusing 5′ end biotinylated gene specific primers. These resultantcapturable nucleic acids can be employed to capture the protein memberof interest, e.g., by contacting the capturable nucleic acids with asource of the protein to be identified, e.g., the cellular/tissueextract or lysate, under protein/nucleic acid binding conditions. Theresultant complexes are then purified, e.g., by Streptavidin coatedbeads, to obtain purified amounts of the protein member for subsequentcharacterization. The above protein purification protocol is merelyrepresentative, as any convenient protocol may be employed. Otherprotocols of interest include, but are not limited to: Gygi, S. P et al.Nat. Biotech. (1999), Vol 17: 994-999; and the like.

[0069] Nucleic Acid Binding Sequence Identification

[0070] In certain embodiments, the binding sequence of the nucleic acidmember may be characterized. In certain embodiments, as described above,the array employed in the subject methods includes a plurality ofmolecular beacon probes that each hybridize to the same target nucleicacid, where the distinct probe members of the plurality differ from eachother by hybridizing to different locations of the target nucleic acidto which they hybridize. In those embodiments where a plurality of suchprobes are present for each target nucleic acid, e.g., it includes atiled set of probes for a given target nucleic acid, some members of theprobe set will give rise to a FRET signal and some will not, asillustrated in FIG. 4. By knowing the sequence of the target nucleicacid, as well as the sequence of the probe regions of the molecularbeacon probes that do and do not give rise to a FRET signal, one canreadily approximate the sequence of the protein binding domain of thetarget nucleic acid which is bound by the protein member of theprotein/nucleic acid binding pair.

[0071] Utility

[0072] The subject methods of identifying protein/nucleic acid bindingpairs can be used in a variety of different applications. Representativeapplications of interest include research applications, where thesubject invention is employed to identify and characterizeprotein/nucleic acid binding pairs. As such, one can employ the subjectinvention to rapidly identify and characterize RNA/protein bindingpairs, single-stranded DNA/protein binding pairs (where the proteinmembers may be involved in DNA replication, repair, recombination,etc.), double-stranded DNA/protein binding pairs (where the proteinmembers may be histones, transcription factors, methylases, polymerases,etc.), telomeric DNA/protein binding pairs, secondary structure (e.g.,Z-DNA, G-quartet DNA, triplex DNA, cruciforms, etc.) assuming nucleicacid/protein binding pairs, etc., in various research applications, suchas elucidation of biochemical pathways, e.g., cellular processes such asreplication, transcription, signaling, etc.

[0073] Systems

[0074] Also provided are systems for use in practicing the subjectmethods. The systems typically include at least the following componentswhich are employed in-practicing the subject methods: (a) a molecularbeacon array; (b) protein labeling reagents, where the label of thelabeling reagent and the label of the molecular beacon probes of thearray make up FRET pair; (c) targets nucleic acid generation reagents;(d) a fluorescent signal detector. Specifics regarding each of theseelements are provided above.

[0075] Kits

[0076] Also provided are kits for use in the subject invention. The kitstypically include a molecular beacon array and at least one proteinlabeling reagent where the labeling reagent includes a fluorescent labelthat is selected to make up a FRET pair with the fluorescent label onthe probes of the molecular beacon array, where FRET pairs are describedabove. In certain embodiments, the kits also include reagents necessaryfor generating the target nucleic acids, e.g., buffers, primers,polymerases, RNA isolation reagents, detergents, etc.

[0077] The various components of the kits may be present in one or morecontainers, each with one or more of the various reagents (sometimes inconcentrated form) utilized in the methods.

[0078] Finally, the kits may further include instructions for using thekit components in the subject methods. The instructions may be printedon a substrate, such as paper or plastic, etc. As such, the instructionsmay be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub-packaging) etc. In other embodiments, the instructionsare present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g., CD-ROM, diskette, etc.

[0079] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL

[0080] I. Preparation of Molecular Beacon Arrays-Molecular Beacon Arraysare Prepared by the Following Methods.

[0081] A. Deposition

[0082] In one method, the individual oligonucleotide sequences labeledwith the appropriate molecular beacons (fluorophore donors andacceptors) are prepared by conventional DNA synthesis on solid supportusing known methods, such as, but not limited to, the phosphoramiditestrategy on CPG. When an additional synthetic moiety is necessary toanchor the sequences to a solid support, it is attached to theoligonucleotide sequence during the chemical synthesis of the individualprobe, either at the 5′ or 3′ end, or in the center of the sequence.Those individually prepared probes are then deposited on solid supports,such as glass, by pulse-jet printing or by mechanical methods involvingthe contact between the solid support and a physical carrier (fiberoptics, pins, etc.) to produce a molecular beacon array. Typically, theglass surface is functionalized prior to deposition with a mono ormultilayer of a coating reactive with a natural or synthetic moietywithin the oligonucleotide being deposited. The glass surface isoptionally treated after deposition to covalently fix the DNA moleculesand/or to inactivate the coating reactive groups that were not used inthe attachment of the DNA sequences.B. In Situ Synthesis

[0083] In another method, the molecular beacon probes are synthesizeddirectly on the solid support, such as glass, in a spatially controlledmanner to achieve the formation of individual features to produce amolecular beacon array. The synthesis is typically performed using thephosphoramidite synthetic methodology and the spatial control isachieved during the coupling step using pulse-jet printing technologiesto deposit the phosphoramidite reagents. Other steps of the DNAsynthesis cycle are performed in a flowcell without spatial control.Alternative methods may include the spatial control of the deblock steputilizing, for instance, light activation strategies using photolabileprotection groups or photogenerated acids and bases. The solid supportis typically functionalized with moieties reactive with the first DNAmonomer coupled to the surface, such as hydroxyls or amino groups. Atthe end of the DNA synthesis, protecting groups, such as of the basesand phosphate groups, are removed under alkaline conditions which do notcleave the DNA probes from the surface.

[0084] The molecular beacon probes sequences are typically anchored tothe surface by their 3′ end, although existing chemistry permits theattachment at any location along the sequence, including the 5′ end. Aspacer is typically used between the molecular beacon DNA sequence andthe attachment point of the probe with the solid support. Typicalspacers include polyethylene glycol phosphates and polynuleotides ofnatural, such as T, and synthetic, such as such a basic, nucleic acidmonomers. The fluorophore acceptor is typically placed between thespacer and the DNA sequence, and the fluorophore donor is typicallyplaced at the other extremity of the DNA sequence. The DNA sequence ofthe first 8 bases and of the last 8 bases are chosen to be complementaryto each other to form, in the absence of DNA target, thethermodynamically favored stem loop. The DNA sequence between theflanking stem sequences can be any sequence of natural and modifiednucleic acids monomer necessary to capture the nuclei acid targets.

[0085] II. Preparation of Nucleic Acid Targets

[0086] Target nucleic acids are prepared using already establishedprotocols (e.g., single stranded and double stranded c-DNA prepared byreverse transcription of mRNA.) Total RNA is prepared by precipitationof nucleic acids from cellular extracts and subsequent DNAsel digestion.cRNA is prepared by the method of U.S. Pat. No. 6,132,997, thedisclosure of which is herein incorporated by reference.

[0087] III. Hybridization of Probe:Target Pair

[0088] Mix 2-5 μg of unlabeled target nucleic acid (see section IIabove) in a total volume of 300 μl of hybridization buffer (e.g., fromAgilent, Palo Alto, Calif.) in a hybridization chamber. Incubate thechamber in a 60° C. rotisserie oven with mixing for a period of 12-17hrs. Dismantle the array from the chamber at room temperature in a lowstringency buffer such as 6×SSPE (containing 0.005% sodium laurylsarcosine) and wash the array in the same buffer composition for 1minute. Transfer the array to a fresh solution of high stringency buffersuch as 0.06×SSPE and wash further for 30 seconds to dissociatenon-specifically bound target molecules.

[0089] IV. Preparation of Labeled Proteins

[0090] This protocol will apply for a library of expressed His taggedproteins. Clone a specific cDNA library (from tissue samples beingcompared) in an appropriate expression vector cassette containing anin-frame histidine tag. Transfect cells (mammalian, bacterial, insectetc. . . . ) with this library allowing for expression of the individualHis-Tagged proteins. Concentrate cells by centrifugation and resuspendin 1 ml of Lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH8.0). Add lysozyme to a final concentration of 1 mg/ml. Incubate on icefor 30 minutes and sonicate mixture to lyse cells. Centrifuge lysate at10,000×g for 30 minutes at 4° C. and collect the supernatant.Equilibrate Ni-NTA spin column with 600 μl of lysis buffer bycentrifuging for˜2 minutes at 700×g. Load an equivalent volume of lysatecontaining the His-tagged proteins onto this pre-equilibrated column andcentrifuge for 2 minutes at 700×g. Wash the column twice with 600 μl ofwash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0) andcentrifuge for 2 minutes at 700×g. Elute the His-tagged proteins twicewith 200 μl of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mMimidazole, pH 8.0) and collect the eluate.

[0091] Label the proteins using the appropriate “protein labeling kit”available from Molecular Probes, Inc. and its associated protocol (e.g.,Fluorescein-EX Protein Labeling Kit”).

[0092] V. Binding Labeled Proteins to Hybridized Array

[0093] Perform labeled protein binding experiments by titrating, foreach molecular beacon array, a specific concentration of purifiedlabeled proteins (2-10 μg) under physiological buffer conditions (50 mMNaH₂PO₄, 100 mM NaCl, 1 mM MgCl₂, 1 mM ZnCl₂, 1 mM CaCl₂, and proteaseinhibitors, pH 7.0-8.0) for a period of 1 hour at 37° C. Gently wash theprotein bound array with the binding buffer (50 mM NaH₂PO₄, 100 mM NaCl,1 mM MgCl₂, 1 mM ZnCl₂, 1 mM CaCl₂, and protease inhibitors, pH 7.0-8.0)for 30 sec.

[0094] It is evident from the above results and discussion that thesubject invention provides a number of advantages over the currentnucleic acid/protein binding pair characterization protocols describedin the Background of the Invention Section, above. Unlike LMPCR/TDPCRwhere sequence information is required to footprint the protein-nucleicacid contacts, the subject microarray based technology outlined in thisinvention has this information built-in (as the sequence of thetranscript/probe attached on the surface). In addition, unlikeLMPCR/TDPCR, this technology is technically less challenging and caneasily be practiced by those with moderate familiarity with microarrays,gene expression profiling and protein expression, purification, andlabeling. Furthermore, one of the major advantages of this invention isthat, unlike LMPCR/TDPCR, this technology is very high throughput andcan identify numerous different protein(s) that bind to differentfeatures (each feature representing a particular transcript). LMPCR andTDPCR are not at all amenable to high throughput analysis and requireseveral days for data processing. As such, the subject inventionrepresents a significant contribution to the art.

[0095] All publications and patent application cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0096] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method of identifying protein/nucleic acidbinding pairs, said method comprising: (a) contacting a molecular beaconarray comprising a plurality of distinct molecular beacon probes with apopulation of target nucleic acids to produce a hybridized molecularbeacon array, wherein each distinct probe of said plurality comprises adifferent probe sequence and all of said probes of said plurality sharea common first fluorescent label; (b) contacting said hybridizedmolecular beacon array with a population of fluorescently labeledproteins to produce a protein bound array, where each member of saidpopulation of fluorescently labeled proteins is labeled with a secondfluorescent label that makes up a FRET pair with said first fluorescentlabel; and (c) detecting any FRET generated signals from said array toidentify protein/nucleic acid binding pairs on said array.
 2. The methodaccording to claim 1, wherein said method further comprises detectingany fluorescent signal from said hybridized array prior to step (b) toidentify target nucleic acids hybridized to said hybridized array. 3.The method according to claim 1, wherein said method further comprisescharacterizing the protein of a protein/nucleic acid binding pairidentified by said method.
 4. The method according to claim 1, whereinsaid method further comprises characterizing the protein bindingsequence of a nucleic acid of a protein/nucleic acid binding pairidentified by said method.
 5. The method according to claim 1, whereinsaid target nucleic acid and fluorescently labeled protein populationsare generated from the same tissue/cellular source.
 6. The methodaccording to claim 1, wherein said target nucleic acid and fluorescentlylabeled protein populations are generated from different tissue/cellularsources.
 7. The method according to claim 1, wherein said hybridizedarray is contacted with two differentially labeled protein populations.8. The method according to claim 7, wherein said two differentiallylabeled protein populations make up a test/control pair.
 9. The methodaccording to claim 8, wherein said two differentially labeled proteinpopulations make up a normal/disease pair.
 10. The method according toclaim 1, wherein said molecular beacon array comprises two or moredistinct molecular beacon probes that hybridize to the same targetnucleic acid, wherein said two or more distinct probes differ from eachother by hybridizing to different locations of said target nucleic acid.11. A system for use in identifying protein/nucleic acid binding pairs,said system comprising: (a) a molecular beacon array comprising aplurality of distinct molecular beacon probes, wherein each distinctprobe of said plurality comprises a different probe sequence and all ofsaid probes of said plurality share a common first fluorescent label;(b) a labeling reagent for labeling a protein population with a secondfluorescent label, wherein said first and second labels make up a FRETpair; (c) reagents for generating a population of target nucleic acids;and (d) a fluorescence detector device.
 12. The system according toclaim 11, wherein said molecular beacon array comprises two or moredistinct molecular beacon probes that hybridize to the same targetnucleic acid, wherein said two or more distinct probes differ from eachother by hybridizing to different locations of said target nucleic acid.13. The system according to claim 11, wherein said system includes twodifferent labeling reagents for producing two differentially labeledprotein populations that are each labeled with a different secondfluorescent labeled that makes up a FRET pair with said firstfluorescent label.
 14. The system according to claim 11, wherein saidfluorescence detector device is a fluorescent scanner.
 15. The systemaccording to claim 11, wherein said system further comprises reagentsnecessary for identifying a protein component of an identifiedprotein/nucleic acid binding pair.
 16. A kit for use in identifyingprotein/nucleic acid binding pairs, said kit comprising: (a) a molecularbeacon array comprising a plurality of distinct molecular beacon probes,wherein each distinct probe of said plurality comprises a differentprobe sequence and all of said probes of said plurality share a commonfirst fluorescent label; (b) a labeling reagent for labeling a proteinpopulation with a second fluorescent label, wherein said first andsecond labels make up a FRET pair; and (c) reagents for generating apopulation of target nucleic acids.
 17. The kit according to claim 16,wherein said molecular beacon array comprises two or more distinctmolecular beacon probes that hybridize to the same target nucleic acid,wherein said two or more distinct probes differ from each other byhybridizing to different locations of said target nucleic acid.
 18. Thekit according to claim 16, wherein said kit includes two differentlabeling reagents for producing two differentially labeled proteinpopulations that are each labeled with a different second fluorescentlabeled that makes up a FRET pair with said first fluorescent label. 19.The kit according to claim 16, wherein said kit further comprisesreagents necessary for identifying a protein component of an identifiedprotein/nucleic acid binding pair.
 20. A substrate comprising a surfacehaving at least one protein/nucleic acid binding pair immobilizedthereon, wherein each protein/nucleic acid binding pair comprises: (a) amolecular beacon probe comprising a first fluorescent label; (b) anunlabeled target nucleic acid hybridized to said molecular beacon probe;and (c) a fluorescently labeled protein labeled with a secondfluorescent label and bound to said unlabeled target nucleic acid,wherein said second fluorescent label and said first fluorescent labelmake up a FRET pair.
 21. The substrate according to claim 20, whereinsaid substrate comprises two or more different protein/nucleic acidbinding pairs immobilized on said surface.
 22. The method according toclaim 1, wherein said method further comprises a data transmission stepin which a result from a reading of the array is transmitted from afirst location to a second location.
 23. The method according to claim22, wherein said second location is a remote location.
 24. A methodcomprising receiving data representing a result of a reading obtained bythe method of claim
 1. 25. A method of identifying protein/nucleic acidbinding pairs, said method comprising: (a) contacting a nucleic acidprobe array comprising a plurality of distinct probe nucleic acids witha population of target nucleic acids to produce a hybridized array,wherein each distinct probe nucleic acid of said plurality comprises adifferent probe sequence; (b) contacting said hybridized array with apopulation of labeled proteins to produce a protein bound array; and (c)detecting any surface bound protein/target nucleic acid complexes toidentify protein/nucleic acid binding pairs on said array.
 26. Themethod according to claim 25, wherein said labeled proteins are labeledwith a first fluorescent label.
 27. The method according to claim 26,wherein said target nucleic acids are labeled with a second fluorescentlabel, wherein said first and second fluorescent labels make up a FRETpair.
 28. The method according to claim 25, wherein said labeledproteins are labeled with an indirectly detectable label.
 29. The methodaccording to claim 25, wherein said method further comprises contactingsaid hybridized array with a second population of labeled proteins thatare distinguishably labeled from said first population of labeledproteins.